Pseudomonas aeruginosa produces the virulence factor, ETA (exotoxin A), which catalyses an ADP-ribosyltransferase reaction of its target protein, eEF2 (eukaryotic elongation factor-2). Currently, this protein–protein interaction is poorly characterized and this study was aimed at identifying the contact sites between eEF2 and the catalytic domain of ETA (PE24H, an ETA from P. aeruginosa, a 24 kDa C-terminal fragment containing a His6 tag). Single-cysteine residues were introduced into the toxin at 21 defined surface-exposed sites and labelled with the fluorophore, IAEDANS [5-(2-iodoacetylaminoethylamino)-1-napthalenesulphonic acid]. Fluorescence quenching studies using acrylamide, and fluorescence lifetime and wavelength emission maxima analyses were conducted in the presence and absence of eEF2. Large changes in the microenvironment of the AEDANS [5-(2-aminoethylamino)-1-naphthalenesulphonic acid] probe after eEF2 binding were not observed as dictated by both fluorescence lifetime and wavelength emission maxima values. This supported the proposed minimal contact model, which suggests that only small, discrete contacts occur between these proteins. As dictated by the bimolecular quenching constant (kq) for acrylamide, binding of eEF2 with toxin caused the greatest change in acrylamide accessibility (>50%) when the fluorescence label was near the active site or was located within a known catalytic loop. All mutant proteins showed a decrease in accessibility to acrylamide once eEF2 bound, although the relative change varied for each labelled protein. From these data, a low-resolution model of the toxin–eEF2 complex was constructed based on the minimal contact model with the intention of enhancing our knowledge on the mode of inactivation of the ribosome translocase by the Pseudomonas toxin.
Abbreviations used: ADPRT, ADP-ribosyltransferase; AEDANS, 5-(2-aminoethylamino)-1-naphthalenesulphonic acid; β-TAD, β-methylene-thiazole-4-carboxamide adenine dinucleotide; DTT, dithiothreitol; eEF2, eukaryotic elongation factor 2; ETA, exotoxin A; IAEDANS, 5-(2-iodoacetylaminoethylamino)-1-napthalenesulphonic acid; MIANS, 2-(4´-maleimidylanilino)naphthalene-6-sulphonic acid.